Illumina HiSeq 2500 sequencing; RNA-seq of primary rat macrophages cultured on substrates of defined stiffness
RNA-seq of primary rat macrophages cultured on substrates of defined stiffness
Macrophages were derived from adult rat bone marrow hematopoietic stem cells.  Hematopoietic stem cells differentiate into macrophages in the presence of macrophage colony stimulating factor present in BMDM. After 6 days to allow differentiation of Hematopoietic stem cells differentiate into macrophages, the medium in the dishes was removed to dispose of any cells which had not attached to the substrate. The remaining cells were washed with warm DPBS followed by CellStripper solution (Corning, 25-056-CI). Cells were incubated in CellStripper for 5 min at 37 ÂºC to detach them from the substrate. The cell suspension was collected and an equal volume of BMDM was added to inactivate the CellStripper solution. Cells were then centrifuged at 1000 rpm for 10 min and the supernatant was removed. Upon re-suspension in BMDM and counting, cells were plated onto polyacrylamide substrates at a density of 10,000 cells/cm2. RNA extraction was carried out at day 3 of culture on polyacrylamide substrates using an RNeasy Plus Micro Kit (Qiagen, 74034). Prior to and at regular intervals during the extraction procedure, work surfaces and pipettes were cleaned with RNase Zap decontamination solution (ThermoFisher, AM9780) to inactivate RNases and prevent sample RNA degradation Library preparation was thereafter carried out using an Ovation RNA-Seq System V2 kit (NuGen, 7102-32), following manufacturer instructions